This invention relates to an anthraquinone and its derivatives, in particular, although not exclusively, including its applications in a range of fluorescence detection technologies.
There are a number of DNA-binding fluorochromes available which cover the UV and visible region of the spectrum. Recently, very bright DNA-intercalating cyanine fluorochromes, based upon modified dimers of thiazole orange, have become commercially available. These cyanine dyes do not share the cell permeant properties of other DNA specific UV-activated fluorochromes. Furthermore, the commonly used DNA-interactive fluorochromes have fluorescent signatures which overlap those of other fluorochromes, activated in the spectral range of visible light, which are used as molecular tags to probe aspects of cell biology or biological structures. Examples of currently known cyanine dyes are disclosed in U.S. Pat. No. 5410030 and U.S. Pat. No. 5436134.
The present invention seeks to develop cell permeant DNA-interactive agents which may provide a fluorescence signature extending in to the infra red region of the spectrum. Such an agent could, for example, be optimally excited by red-line emitting lasers in multi-laser/multi-fluorochrome applications for both fixed specimens and viable cells.
Thus, in accordance with a first aspect of the present invention, there is provided a compound of the following formula (I): 
wherein each of X1 and X2 are independently NHxe2x80x94Axe2x80x94NR1R2, and wherein A is a C2-8 alkylene group and R1 and R2 are independently selected from hydrogen, C1-4 alkyl, C2-4 hydroxyalkyl and C2-4 aminoalkyl, or R1 and R2 together form a C2-6 alkylene group which with the nitrogen atom to which R1 and R2 are attached forms a heterocyclic ring, or an N-oxide derivative thereof, and wherein the compound (I) or its N-oxide derivative is optionally in the form of an acid salt derived from an organic or inorganic acid.
The term xe2x80x9calkylenexe2x80x9d here is used to mean an alkyl chain.
In a preferred embodiment, when R1 and R2 form a heterocyclic ring, the ring has 3 to 7 carbon atoms therein. Preferably, both X1 and X2 are both NH(CH2)2NR1R2. In particular, it is preferred that R1 and R2 are both C1-4 alkyl groups, preferably methyl groups.
According to a second aspect of the present invention, there is provided a compound of the following formula (II): 
In one embodiment, compound (II) may be in the form of its N-oxide derivative.
The compound of the general formula (I) and, in particular the specific compound (II) may be used as, for example, a DNA dye and may be a pure synthetic compound which is soluble in biologically compatible solvents including water. Compound (II) has a high infinity for DNA (the DNA binding constant is approximately 10e7 M-1) and has the capacity to enter living cells rapidly.
The absorbance spectrum for compound (II) shows Exxcexmax near 647 nm and produces a fluorescence spectrum extending from 665 nm out to beyond 780 nm wavelengths (Emxcexmax is about 677.5 nm).
According to a further aspect of the present invention, there is provided a method of preparing a compound of the following formula (I): 
wherein each of X1 and X2 are independently NHxe2x80x94Axe2x80x94NR1R2, and wherein A is a C2-8 alkylene group and R1 and R2 are independently selected from hydrogen, C1-4 alkyl, C2-4 hydroxyalkyl and C2-4 aminoalkyl, or R1 and R2 together form a C2-6 alkylene group which with the nitrogen atom to which R1 and R2 are attached forms a heterocyclic ring, or an N-oxide derivative thereof, and wherein the compound (I) or its N-oxide derivative is optionally in the form of an acid salt derived from an organic or inorganic acid,
the method comprising the step of reacting a compound of the following formula (III) 
with NH2xe2x80x94Axe2x80x94NR1R2, wherein A, R1 and R2 are as defined above.
The method preferably further comprises the step of treating the resultant compound with an acid, preferably concentrated sulphuric acid. In addition, in a preferred embodiment, the method may further comprise subsequent treatment with sodium chlorate and/or sodium hydrogen sulphite.
Modelling has demonstrated that the compounds of the present invention can form stable, intercalated complexes with DNA. Thus, according to a further aspect of the present invention, there is provided a fluorescent complex comprising a nucleic acid and a compound of the following formula (I): 
wherein each of X1 and X2 are independently NHxe2x80x94Axe2x80x94NR1R2, and wherein A is a C2-8 alkylene group and R1 and R2 are independently selected from hydrogen, C1-4 alkyl, C2-4 hydroxyalkyl and C2-4 aminoalkyl, or R1 and R2 together form a C2-6 alkylene group which with the nitrogen atom to which R1 and R2 are attached forms a heterocyclic ring, or an N-oxide derivative thereof, and wherein the compound (I) or its N-oxide derivative is optionally in the form of an acid salt derived from an organic or inorganic acid.
The nucleic acid is preferably DNA. It has been found that the DNA may be present in a living cell. The compounds of the present invention may stain fixed human chromosomes. As the DNA:Compound molar ratio increases there is a bathochromic shift in the compound plus DNA solution spectrum. At high DNA:Compound ratios, attainable within living cells, the spectral shift contributes to an already significant separation of the compound-DNA emission spectrum from that of an example of a red-fluorescing compound Cy 5.
According to a further aspect of the present invention, there is provided a method of analysing a cell or biological material containing one or more nucleic acids, comprising the steps of:
a) preparing a biologically compatible solution containing a compound of the formula (I): 
wherein each of X1 and X2 are independently NHxe2x80x94Axe2x80x94NR1R2, and wherein A is a C2-8 alkylene group and R1 and R2 are independently selected from hydrogen, C1-4 alkyl, C2-4 hydroxyalkyl and C2-4 aminoalkyl, or R1 and R1 together form C2-6 alkylene group which with the nitrogen atom to which R1 and R2 are attached forms a heterocyclic ring, or an N-oxide derivative thereof, and wherein the compound (I) or its N-oxide derivative is optionally in the form of an acid salt derived from an organic or inorganic acid;
b) treating the cell or biological material with the biologically compatible solution;
c) exciting the compound (I) in the treated cell or biological material with a light source; and
d) detecting the emitted fluorescence signal.
The compound of formula (I) may be present in its free state or be complexed to other molecule(s), for example either by covalent or non-covalent attachment.
The light source preferably provides wavelength(s) in the spectral region of the wavelength(s) of maximum absorption of compound (I).
It has been found that the fluorescence signature of the compounds of the present invention extends to the infra red region of the spectrum. The compound of the present invention may be present in the cell or biological material in combination with one or more other fluorochromes or light-emitting compounds. The other fluorochromes may emit in the UV or visible region of the spectrum. Thus, the compounds of the present invention lend themselves to multi-parameter analysis with other fluorochromes with spectra which overlap with those of the commonly used visible-region DNA probes.
The one or more other compounds may be used, for example, to detect Annexin V and is preferably used in combination with the N-oxide derivative of compound (I). Flow cytometric analysis, for example with the instrument in dual laser mode, may be used. The invention thus may provide a way of discriminating intact viable cells from those undergoing the various stages of cell death.
Thus, the compounds of the present invention provide far red/infra red fluorescent permeant DNA dyes suitable for cellular DNA analysis where intact cells may be required, for example the detection of molecules either on the cell surface (e.g. a receptor molecule or marker for differentiation) or within cells (e.g. cytosolic enzymes) by methods which require the maintenance of membrane integrity to prevent perturbation or loss of such molecules.
As mentioned above, in this method, the compounds of the present invention may stain nucleic acids in fixed human chromosomes, fixed cells and fixed biological materials, and in procedures which modify the permeability of living cell membranes.
According to a further aspect of the present invention, there is provided the use of compound (I) in a biological assay. Compound (I) may be present either in its free state or complexed to other molecules by either covalent or non-covalent attachment in the biological assay. Compound (I) may be present as an N-oxide derivative thereof. The biological assay is preferably a rapid and/or large capacity handling procedure. The use of the compounds of the present invention, as indicated by compound (I), as a discriminating or orientating parameter for cell nuclei has been demonstrated for both flow cytometry and confocal laser scanning microscopy.
In accordance with a further aspect of the present invention, there is provided the use of compound (I) in cytometry. Compound (I) is optionally present as an N-oxide derivative thereof. The cytometry process may be, for example, single beam or multi-beam flow cytometry.
By way of example, single beam (488 nm) flow cytometry has been used to demonstrate the utility of compound (I)-nuclear DNA fluorescence (preferably compound (II)-nuclear DNA fluorescence) as a discriminating parameter for human blood and lymphoma cells, in combination with fluorochrome-labelled antibodies for the detection of surface antigens and subpopulation recognition. Compound (I) fluorescence was found to reflect cellular DNA content as evidenced by cell cycle DNA distribution profiles for exponentially proliferating cell populations showing a steady-state or asynchronous distribution of cells with respect to cell cycle age, or for perturbed cell populations in which, for example, drug action has caused the delay or arrest of cells at a given point in the cell cycle. In one embodiment, dual beam (488 nm/633 nm) flow cytometry shows the selective excitation of compound (I), preferably compound (II), and fluorescein in intact cells. In addition, in one embodiment, the application of compound (I), preferably compound (II), in triple beam flow cytometry (multiline UV/488 nm/633 nm) has been demonstrated in applications involving delayed signal discrimination where beam separation allows for the discrimination of the excitation beam associated with a fluorescence emission signal by reference to the delay in signal arrival at a detector.
According to a further aspect of the present invention, there is provided the use of compound (I) in microscopy. Compound (I) may be present as its N-oxide derivative. Preferably the microscopy is confocal laser scanning microscopy (CLSM). By way of example, CLSM employing either 647 nm or 568 nm wavelength excitation of intracellular compound (I), preferably intracellular compound (II), shows fluorescence specifically located in the nucleus revealing nuclear architecture within living or fixed human cells.
According to a further aspect of the present invention, there is provided the use of compound (I) as a nuclear staining agent. Compound (I) may be present as its N-oxide derivative.
According to a further aspect of the present invention, there is provided the use of compound (I) as an imaging agent. Compound (I) may be present as its N-oxide derivative.
In one embodiment, compound (I) can be used as an imaging agent in multi-photon excitation imaging.
Dual wavelength imaging, using compound (I) to reveal nuclear form, may be used to demonstrate the heterogeneity in esterase-dependent fluorescein loading of whole cells and in the assessment of mitochondrial function by rhodamine 123 labelling. In such imaging applications, compound (I) shows no evidence of photo bleaching and was persistent.
Thus, the compounds of the present invention can be considered as a fluorochrome for application as an agent in the use, calibration, standardization, and configuration of fluorescence-based systems. The preferred compound of the present invention is compound (II)-deep red fluorescing bisalkylaminoanthraquinone (DRAQ5).
It has been found that the high penetration of red line laser beams into tissues and the permeant properties of the compounds of the present invention provide a combination which allows three dimensional orientation and location of nuclei within living tissues. In addition, the availability of low cost HeNe lasers or other red light-emitting devices with enhanced power enables the compounds of the present invention to find applications in detection systems where their fluorescence signature can be used as a discriminating parameter.
Whilst the invention has been described above, it extends to any inventive combination of the features set out above or in the following description.